1. Field of the Invention
This invention relates to antibodies modified with a cytotoxic substance, which may be used to treat chronic disorders in humans induced by viral infections, for example, AIDS viruses and leukemia viruses.
2. Description of Prior Art
Nowadays, certain chronic disorders in humans, for example, Acquired Immunodeficiency Syndrome (AIDS), AIDS-Related Complex (ARC) and adult T-cell leukemia, which are induced by the replication of viruses in the body of human hosts, are well recognized as world-wide epidemics.
It is also known that human immunodeficiency virus (HIV), an etiological factor for such disorders is a human retrovirus.
As is well known, prototype HIV are human T-lymphotropic virus type III (HLTV-III) and lymphadenopathy associated virus (LAV), and human T-cell leukemia virus I (HTLV-I) is pathogenic for leukemia and immunodeficiency syndrome.
For example, the most profound hematologic features associated with AIDS are the functional impairment and quantitative depletion of the helper/inducer subset of T-lymphocytes which express the CD4 surface antigen. HIV-induced immunosuppression results in a variety of deficiencies of the host defense system. The immune defect appears to be progressive and irreversible and results in a very high mortality rate.
In the first stage of HIV infection to T cells, cell-free infection viz. attachment of cell-free virons to the target receptor CD4 antigen occurs. However, HIV may also spread by cell-to-cell infection viz. by fusion of infected T cells with uninfected T cells so that the formation of syncytia (polynucleated giant cells) occurs in organs such as the brain and the lymph nodes. The depletion of CD4-positive cells may occur because the HIV-infected T cells are susceptible to the cytopathic effects of HIV.
Another feature of such chronic disorders induced by infection of viruses resides in the fact that the incubation period is very long. It is known that HIV infects not only the helper/inducer subsets of T cells but also the cells of the monocytes/macrophage lineage. It is also known that, in such cases, most of monocytes/macrophages and certain T cells are resistant to the cytopathic effects of HIV and are thus considered to act as the reservoir cells of the viruses.
It is further known that polyclonal antibodies against HIV are present in blood obtained from HIV-infected humans, but the neutralizing activities of such antibodies are, in general, very weak. Thus, even though at the initial stage of the infection, cells infected with the viruses may more or less be killed in the body of the hosts, the immunodeficiency of the hosts gradually falls and eventually the host will die.
The existence of certain structural antigens of HIV including core (gag) antigens and envelope antigens is also known. The viral envelope comprises a 160 kilodalton (gp160) precursor glycoprotein which is subsequently cleaved into 120 kd (gp120) and 41 kd (gp41) glycoproteins present in the viral particles. The external envelope protein of HIV gp120 is the most important glycoprotein with respect to the following characteristics:
(1) Gp120 and/or certain fragments of gp120 are capable of inducing polyclonal neutralizing antibodies in experimental animals. This means that gp120 is at least one of the target molecules of neutralizing antibodies [as disclosed, for example, in Lasky, L. A. et al, Science, 233, 209-212 (1986); Robbey, W. G. et al, Proc. Natl. Acad. Sc. U.S.A., 83, 7023-7027 (1986) and Putney S. D. et al., Science, 234, 1392-1395 (1987). PA0 (2) The infection of HIV is initiated by binding of gp120 to the receptor CD4 molecule. This means that gp120 is a critical molecule for HIV with respect to the infection to target cells [as disclosed, for example, in McDougal J. S. et al, Science, 231, 382-385 (1986)]. PA0 (3) The formation of syncytia induced by HIV viz. the cell-to-cell infection of HIV depends on the direct interaction of gp120 with CD4 molecules of the uninfected cells [as disclosed, for example, in Lifson J. D. et al, Nature 323, 725-728 (1986)].
In the case of human T-cell leukemia viruses, it is said that gp46, a glycoprotein antigen on the envelope of HLTV-1 corresponding to gp120 of HIV, represents an important etiological factor.
Various monoclonal antibodies against the protein components of HTLV-III or LAV have hitherto been proposed, as exemplified by those against p24, one of the core antigens present on the inside of the viruses [Veronese F. D., Proc. Natl. Acad. Sci. USA., 82, 5199-5202 (1985): those against the product from the poll gene capable of cording the reverse transcriptase of the viruses [Veronese F. D. et al., Science 231, 1289-1291 (1986); and those against gp41, part of the envelope [Veronese F. D. et al., Science 229, 1402-1405 (1985)]. However, none of the known monoclonal antibodies are capable of reacting with gp120 antigen which is important to treat HIV and to protect against HIV infection and are also capable of neutralizing HIV.
Almost all antiviral agents which have ever been proposed to prevent and/or treat HIV infection appear to act as agents to inhibit HIV-specific enzymes. Thus, for example, azidothymidin and dideoxycytidin are agents to inhibit reverse transcriptase, and castanospermin is an agent to inhibit modification of viral proteins.
Even though these agents are more or less capable of inhibiting the infection of fresh viruses just produced in the body of the patients to uninfected cells, it is difficult to positively kill the cells already infected.
On the other hand, various attempts have been made to specifically kill tumour cells by using antibodies conjugated with a substance which is toxic against the tumour cells (the so-called immunotoxin) [for example, E. S, Vietta et al, Cell, vol. 41, 653-654 (July 1986); and I. Pastan et al, Cell, vol. 47, 641-648 (December 1986)].
It has also been proposed to use monoclonal antibodies conjugated with particles capable of emitting .alpha.-rays which are toxic against tumour cells [R. M. Macklis et al, Science vol. 240, 1024-1027 (20 May 1988)]. However, there are still serious problems to be solved in this regard. For example, it is not yet clear whether an antibody Capable of specifically reacting with antigens of tumour cells really exists.
With regard to treatment of chronic disorders induced by viral infections, such an immunotoxin has not yet been proposed by various reasons. Clearly one of the main reasons resides in that any antiviral antibodies which may effectively be used for this purpose has not yet been proposed. Thus, a toxic substance which may advantageously be used for this purpose has not yet been clarified.
The present inventor has proposed a monoclonal antibodies designated as 0.5.beta. antibody having the following characteristics:
(a) capable of substantially neutralizing human immunodeficiency viruses (HIV) by binding to a glycoprotein antigen having a molecular weight of about 120,000, located on the envelope of said viruses; PA1 (b) classified into IgG.sub.1 ; PA1 (c) capable of inhibiting the formation of syncytia between the cells infected with human T-lymphotropic viruses III and uninfected cells by binding to the surfaces of the infected cells; PA1 (d) capable of binding to the precursor of a glycoprotein antigen of HIV, having a molecular weight of about 160,000 dalton; and PA1 (e) capable of recognizing an epitope located within a range of Nos. 308-331 of the amino acid sequence of gp120 antigen of human immunodeficiency viruses [measured by the method of Ratner et al. (Nature, 313, 77-284 (1985)]. PA1 (a) capable of specifically reacting with a glycoprotein having a molecular weight of about 120,000 dalton and located on the envelope of HIV; PA1 (b) classified into IgG.sub.1 ; PA1 (c) capable of inhibiting the formation of syncytia between the cells infected with HTLV-III and uninfected cells; PA1 (d) capable of binding to the precursor of a glycoprotein antigen of HIV, having a molecular weight of about 160,000 dalton: PA1 (e) capable of recognizing an epitope located within the range of Nos. 308-331 of the amino acid sequence of gp120 antigen of HIV; thereby to result in an antibody or fragment thereof capable of substantially inhibiting the growth of the human cells infected with said virus and neutralizing said infected cells.
This monoclonal antibody is disclosed in the parent patent application Ser. No. 198,957 filed in the name of the present inventor.
Although 0.5.beta. antibody is capable of effectively reacting with gp120 of HIV and neutralizing the viruses, it may be difficult to effectively inhibit the growth of the cells infected with the viruses viz. the cells capable of producing the viruses.
The present invention is based upon the discovery that it is possible to obtain an antibody capable of neutralizing the viruses and also capable of inhibiting the growth of the cells infected with the viruses by modifying 0.5.beta. antibody with certain substances.